The protease inhibitor E64d might attenuate the development of experimental anti-glomerular basement membrane disease through regulating the activation of Th1 cells

Article

作者: Cao, Xin ; Cheng, Xi ; Gu, Qiu-Hua ; Xu, Hao ; Jia, Jun-Ya ; Yan, Tie-Kun

BACKGROUNDCathepsins have been recently identified as a regulator in the activation of Th1 and Th17 cells, which play an important role in the pathogenesis of anti-glomerular basement membrane (GBM) disease. Whether cathepsins contribute to the development of anti-GBM disease through regulating the activation of CD4⁺ T cell is still unclear.METHODSRats with experimental anti-GBM disease was established by immunization with the nephritogenic T cell epitope α3_127-148. E64d, a cysteine cathepsin inhibitor, was administered in vitro and vivo to evaluate the effect of cathepsins on regulating the activation of antigen specific T cells and the development of anti-GBM disease.RESULTSIn rats with experimental anti-GBM diseases, E64d treatment not only reduced the levels of proteinuria, serum creatinine and anti-GBM antibody, but also ameliorated the kidney injury with less glomerular IgG deposition, a lower percentage of crescents and less infiltration of CD4⁺ T cells, CD8⁺ T cells and macrophages, as well as a lower percentage of splenic Th1 cells. In vitro, E64d treatment could significantly reduce the production of IFN-γ in the supernatant which might be produced by the activation of Th1 cells after being recalled with the autoantigen α3_127-148. We also found the CD4⁺ T cells of rats with anti-GBM disease had an increased expression of cathepsin L (Cts-L), and the percentage of CD4⁺ T cells with extracellular expression of Cts-L was obviously higher, indicating it as a potential key regulator.CONCLUSIONSE64d might attenuate the development of anti-GBM disease by participating in the activation of Th1 cells, indicating it as a potential drug for anti-GBM disease in the future.

2023-12-01·Microbial Pathogenesis

Clostridioides difficile PCR ribotypes 001 and 084 can trigger autophagy process in human intestinal Caco-2 cells

Article

作者: Zali, Mohammad Reza ; Amirkamali, Sahar ; Klionsky, Daniel J ; Azimirad, Masoumeh ; Noori, Maryam ; Asadzadeh Aghdaei, Hamid ; Yadegar, Abbas ; Nasiri, Gelareh

Autophagy is a homeostatic process that can promote cell survival or death. However, the exact role of autophagy in Clostridioides difficile infection (CDI) is still not precisely elucidated. Here, we investigate the role of distinct C. difficile ribotypes (RTs) in autophagy induction using Caco-2 cells. The expression analysis of autophagy-associated genes and related miRNAs were examined following treatment of Caco-2 cells with C. difficile after 4 and 8 h using RT-qPCR. Toxin production was assessed using enzyme-linked immunosorbent assay (ELISA). Immunofluorescence analysis was performed to detect MAP1LC3B/LC3B, followed by an autophagic flux analysis. C. difficile significantly reduced the viability of Caco-2 cells in comparison with untreated cells. Elevated levels of LC3-II and SQSTM1/p62 by C. difficile RT001 and RT084 in the presence of E64d/leupeptin confirmed the induction of autophagy activity. Similarly, the immunofluorescence analysis demonstrated that C. difficile RT001 and RT084 significantly increased the amount of LC3-positive structures in Caco-2 cells. The induction of autophagy was further demonstrated by increased levels of LC3B, ULK1, ATG12, PIK3C3/VPS34, BECN1 (beclin 1), ATG5, and ATG16L1 transcripts and reduced levels of AKT and MTOR gene expression. The expression levels of MIR21 and MIR30B, microRNAs that suppress autophagy, were differentially affected by C. difficile. In conclusion, the present work revealed that C. difficile bacteria can induce autophagy through both toxin-dependent and -independent mechanisms. Also, our results suggest the potential role of other C. difficile virulence factors in autophagy modulation using intestinal cells in vitro.

2023-10-01·3 Biotech

Understanding the role of conserved proline and serine residues in the SARS-CoV-2 spike cleavage sites in the virus entry, fusion, and infectivity

Article

作者: Lohiya, Bharat ; Samal, Sweety ; Maithil, Vikas ; Kaur, Gurleen ; Khatri, Ritika ; Asthana, Shailendra ; Goswami, Abhishek ; Sarmadhikari, Debapriyo

The spike (S) glycoprotein of the SARS-CoV-2 virus binds to the host cell receptor and promotes the virus's entry into the target host cell. This interaction is primed by host cell proteases like furin and TMPRSS2, which act at the S1/S2 and S2´ cleavage sites, respectively. Both cleavage sites have serine or proline residues flanking either the single or polybasic region and were found to be conserved in coronaviruses. Unravelling the effects of these conserved residues on the virus entry and infectivity might facilitate the development of novel therapeutics. Here, we have investigated the role of the conserved serine and proline residues in the SARS-CoV-2 spike mediated entry, fusogenicity, and viral infectivity by using the HIV-1/spike-based pseudovirus system. A conserved serine residue mutation to alanine (S2´S-A) at the S2´ cleavage site resulted in the complete loss of spike cleavage. Exogenous treatment with trypsin or overexpression of TMPRSS2 protease could not rescue the loss of spike cleavage and biological activity. The S2´S-A mutant showed no significant responses against E-64d, TMPRSS2 or other relevant inhibitors. Taken together, serine at the S2´ site in the spike protein was indispensable for spike protein cleavage and virus infectivity. Thus, novel interventions targeting the conserved serine at the S2´ cleavage site should be explored to reduce severe disease caused by SARS-CoV-2-and novel emerging variants.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13205-023-03749-y.

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适应症	最高研发状态	国家/地区	公司	日期
抗肾小球基膜疾病	临床前	中国	天津医科大学总医院	2024-03-10
阿尔茨海默症	临床前	美国	American Life Science Pharmaceutical, Inc.	2013-07-01
RNA病毒感染	药物发现	德国	Deutsches Elektronen-Synchrotron DESY	2024-04-17